It has been proposed that adenosine, a product of normal cellular metabolism, may be an important regulator of coronary blood flow and cardiac function. However, direct confirmation of this hypothesis awaits precise measurements of instantaneous interstitial adenosine concentrations. In this application, a new approach for direct determination of interstitial adenosine concentration is proposed. Preliminary experiments indicate that isolated rat hearts, perfused at constant high flow with colloid-free, physiological salt solution, are continually filtering fluid out of the vascular bed. A portion of this fluid percolates through the interstitium and is exuded at the epicardial surface of the heart. In preliminary work, we have developed techniques for collecting this fluid and measuring its adenosine and inosine concentration. Under control conditions, surface exudate adenosine ([ADO]i) was 141 plus/minus 14 nM while the adenosine concentration in the venous effluent ([ADO]v) was 29 plus/minus 4 nM (N = 22). Inosine levels were 1.4 to 1.8-fold higher than adenosine levels. Increasing metabolic activity (by rapid pacing or isoproterenol infusion) initially increased both adenosine and inosine levels in surface exudate and venous effluent. The proposed method will provide rapid assessment of [ADO]i and allow us to determine to what extent changes in [ADO]i influence cardiac function and [ADO]v under various physiological conditions. Initial experiments are designed to fully characterize this preparation. Subsequent experiments will be performed to address the following specific questions: 1) What are the true concentration-response relationship between [ADO]i and cardiac variables such as coronary vascular resistance, ventricular beating rate, action potential conduction velocity and contractility? 2) Do imbalances between the oxygen supply and oxygen demand of the myocardium result in alterations in [ADO]i? 3) Are the changes in [ADO]i resulting from any given metabolic imbalance large enough to produce the observed alterations in cardiac function? 4) What is the relationship between adenosine and inosine levels in the fluid samples under various testing conditions? and 5) What are the characteristics of the capillary endothelial adenosine barrier in this preparation? These studies will provide important information about levels of interstitial adenosine and how adenosine might influence coronary blood flow and cardiac function in normal and abnormal states.